CN105717035B - Flow cytometry device and method - Google Patents
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
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Abstract
The invention proposes a kind of purposes of Flow cytometry device and method and binary optical device in Flow cytometry or preparation Flow cytometry device.The Flow cytometry device includes: that hot spot forms component, and the hot spot forms component and is configured as being suitable for forming at least two hot spots on F-L curve.The Flow cytometry device that the embodiment of the present invention is proposed can be realized the Accurate Determining to biological particle speed, single particle resolving power and, testing result stability height strong to the detection power of small-signal.
Description
Technical field
The present invention relates to micro-optics detection technique fields, in particular it relates to Flow cytometry device, streaming
The use of cell art detection method and binary optical device in Flow cytometry or preparation Flow cytometry device
On the way.
Background technique
Flow cytometry can be used for detecting by the cell of fluorescent marker, embryo, RNA, DNA, protein particulate, micro- life
The biological particles such as object, virus.In detection, flow cytometry needs are radiated at by means of the laser facula Jing Guo shaping positioned at liquid stream
On the biological particle at center, biological particle issues scattering light and fluorescence under the excitation of laser facula.These scattering light and fluorescence
Signal is collected by electrooptical devices such as photomultiplier tube, photodiodes, is analyzed using detection system, to obtain biology
The physics and chemical information of particle, such as cell size, surface granularity, antigenic information.Testing principle based on flow cytometry,
The shape and quality of laser facula are to influence detection sensitivity, the key factor of single particle resolving power.
Currently, commercial flow cytometer is generally shaped as gauss laser using two cylindrical lens the Gauss light of ellipse
Spot, the short axle of elliptical spot be parallel to the flow direction of biological particle, long axis perpendicular to biological particle flow direction, with improve to single
The resolving power of biological particle prevents missing inspection to biological particle simultaneously.
However, Flow cytometry device still has further exploitation and improves.
Summary of the invention
The application is to be proposed based on inventor to following problems and true discovery:
At present commercialization flow cytomery stability and the resolving power of single biological particle is difficult to further increase,
It is difficult to realize high speed and high-throughput detection, and is unable to satisfy the requirement of testing the speed to biological particle.Inventors discovered through research that
The key of the above problem is caused to be in commercial flow cytometer elliptical spot at present that photic-energy transfer is uneven, biological particle is logical
When crossing the relative position of hot spot and changing, the optical signalling of output can change therewith, and ellipse light spot is Gauss
When distribution, the obscure boundary of hot spot is clear, meanwhile, single hot spot is unable to satisfy the real time speed measuring requirement to each biological particle.
The present invention is directed to solve at least to a certain extent it is above-mentioned in the related technology the technical issues of one of.For this purpose, this hair
Bright propose using the binary optical device for having surface micro-structure realizes spot shaping, and then tool is realized on output plane
Have the hot spot of following features: hot spot boundary is sharp keen, and luminous energy is uniformly distributed in hot spot, can be realized the high pass measurement to biological particle
It is fixed；Hot spot is made of two or more figures, can be realized the speed the real time measure to each biological particle；Hot spot can be by multiple
Various sizes of figure composition, can take into account single particle resolving power and Detection of Weak Signals.
In turn, in the first aspect of the present invention, the invention proposes a kind of Flow cytometry devices.According to the present invention
Embodiment, comprising: hot spot forms component, and the hot spot forms component and is configured as being suitable for forming at least two on F-L curve
A hot spot.The Flow cytometry device that the embodiment of the present invention is proposed can be realized the accurate survey to biological particle speed
It is fixed, single particle resolving power and strong to the detection power of small-signal.
According to an embodiment of the invention, above-mentioned Flow cytometry device can further include following supplementary technology
At least one feature:
According to an embodiment of the invention, it is binary optical device that the hot spot, which forms component,.Binary optical device makes this
The detection stability of the Flow cytometry device of inventive embodiments, single particle resolving power and to Detection of Weak Signals ability into
One step improves.
According to an embodiment of the invention, at least two hot spot is separately rectangle.Rectangular light spot makes this hair
The detection stability of the Flow cytometry device of bright embodiment, single particle resolving power and high speed and high-throughput detection efficiency into
One step improves.
According to an embodiment of the invention, at least two spot separations setting, and the central axes of the rectangle are located at
On the F-L curve.The setting of above-mentioned rectangular light spot is so that the detection sensitivity of cell art detection device further increases.
According to an embodiment of the invention, the long side length of at least two hot spot is 10~100 microns, it is described at least
The bond length of the both ends hot spot of two hot spots is the size determination based on sample to be tested.The long side length of rectangular light spot is 10
~100 microns of missing inspections that can be avoided to greatest extent to biological particle sample.The bond length of both ends hot spot is based on to test sample
What the size of product determined, it can further improve the resolving power to single biological particle sample.
According to an embodiment of the invention, the both ends hot spot of at least two hot spot is respectively provided with identical size, and
And the long side length of at least two hot spot is equal.In turn, the flow cytometry detection device of the embodiment of the present invention is at data
The accuracy of test can be improved in reason by data processing methods such as weighted averages.
According to an embodiment of the invention, the amplitude variance of at least two hot spot is less than 10%, the light intensity of hot spot is equal.
In turn, the flow cytometry detection device of the embodiment of the present invention can be mentioned in data handling by data processing methods such as weighted averages
The accuracy of height test.
According to an embodiment of the invention, the uniformity of the light intensity of at least two hot spots is higher than 80%, light distribution is uniform, into
One step improves the accuracy and sensitivity of Flow cytometry device.
According to an embodiment of the invention, the Flow cytometry device includes: the optical path along incident laser, successively
It is provided with laser, beam expanding lens, the hot spot and forms component, the first aperture and the first condenser lens；Along forward scattering
The optical path of laser is disposed with light barrier, the first optical filtering and the first photoelectric sensor；The laterally light of scattering laser
Road is disposed with the second optical filtering and the second photoelectric sensor；Along the optical path for the fluorescence that is excited, it is poly- to be disposed with second
Focus lens and multiplex fluorescence detection device, wherein the multiplex fluorescence detection device include dichroscope, second orifice diaphragm,
Third optical filtering and third photoelectric sensor；And along observation optical path, be disposed with image optical flame detector, the 4th optical filtering and
Tertiary focusing lens.The Flow cytometry device of the embodiment of the present invention is easy to operate under said modules composition, detection
Stability, accuracy, single particle resolving power and high speed and high-throughput detection efficiency further increase.
In the second aspect of the present invention, the invention proposes a kind of flow cytometry assays.Reality according to the present invention
Apply example, the detection method includes: that at least two hot spots are formed on F-L curve, wherein at least two hot spot be as
Defined in preceding.The flow cytometry assays that the embodiment of the present invention is proposed can Accurate Determining biological particle sample to be tested
The stability of speed, testing result is high, can take into account single particle resolving power and the detection to small-signal.
According to an embodiment of the invention, above-mentioned flow cytometry assays can further include following supplementary technology
At least one feature:
According to an embodiment of the invention, the method is carried out using mentioned-above Flow cytometry device.
As previously mentioned, the Flow cytometry device of the embodiment of the present invention has testing result stability height, the resolution to single particle
Power is strong and the feature strong to the detectability of small-signal, and then the detection method of the embodiment of the present invention is implemented using the present invention
The Flow cytometry device of example detects biological particle sample to be tested, the stability of testing result, to single particle
Resolving power and the detectability of small-signal is further increased.
In the third aspect of the present invention, the invention proposes binary optical devices in Flow cytometry or preparation streaming
Purposes in cell art detection device.According to an embodiment of the invention, binary optical device apply in Flow cytometry or
Prepare Flow cytometry device, the detection method or device can Accurate Determining biological particle speed, to biological particle
The stability of testing result is high, the resolving power to single particle and the detectability height to small-signal.
According to an embodiment of the invention, above-mentioned binary optical device is in Flow cytometry or prepares flow cytometry inspection
The purposes surveyed in device can further include at least one following additional technical feature:
According to an embodiment of the invention, the binary optical device is modulated the phase of incident laser.Utilize binary
Optical device is modulated the phase of incident laser, further increases to the velocity determination accuracy of biological particle, to biology
The stability of the testing result of particle and the resolving power to single particle and the detectability to small-signal further increase.
According to an embodiment of the invention, the material of the binary optical device is selected from quartz, glass, poly-methyl methacrylate
At least one of ester.It selects above-mentioned at least one of material to prepare binary optical device, it is thin to can further improve the streaming
The stability and sensitivity of the detection of born of the same parents' art or Flow cytometry device.
According to an embodiment of the invention, the laser includes selected from least one of Gauss light, planar light and multimode light.Root
According to the embodiment of the present invention, suitable incident laser and its corresponding binary optical device are selected, can further significantly improve institute
State the stability and sensitivity of Flow cytometry or Flow cytometry device.
According to an embodiment of the invention, when the laser is the Gauss light that diameter is 3mm, the binary optical device
Structure is square, and the side length of the square is 3mm and inside has 128*128 step.The embodiment of the present invention it is above-mentioned
The structure of binary optical device further improves the steady of the flow cytometry assays or Flow cytometry device
The qualitative and resolving power to single particle and the detectability to small-signal.
Detailed description of the invention
Fig. 1 is the schematic diagram of double square hot spot according to an embodiment of the present invention；
When Fig. 2 is Flow cytometry according to an embodiment of the present invention, double square hot spot, biological particle and runner it
Between positional diagram；
Fig. 3 is that biological particle according to an embodiment of the present invention passes through the schematic diagram of the optical signal issued when double square hot spot；
Fig. 4 is the phase diagram of one according to an embodiment of the present invention specific binary optical device；
Fig. 5 is the output light distribution map according to an embodiment of the present invention obtained using binary optical device；
Fig. 6 is the schematic diagram of three rectangular light spot according to an embodiment of the present invention；
Fig. 7 is the positional diagram between three rectangular light spots, biological particle and the runner implemented according to the present invention；
Fig. 8 is that biological particle according to an embodiment of the present invention passes through the schematic diagram of the optical signal issued when three rectangular light spots；
Fig. 9 is the phase diagram of one according to an embodiment of the present invention specific binary optical device；
Figure 10 is the output light distribution map according to an embodiment of the present invention obtained using binary optical device；
Figure 11 is the index path of flow cytometry detection device according to an embodiment of the present invention；And
Figure 12 is the structural schematic diagram of Flow cytometry device according to an embodiment of the present invention.
Specific embodiment
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.
Flow cytometry device
In the first aspect of the present invention, the invention proposes a kind of Flow cytometry devices.Reality according to the present invention
Apply example, comprising: hot spot forms component, and the hot spot forms component and is configured as being suitable for forming at least two light on F-L curve
Spot.Reference by location Fig. 1 of F-L curve.At least two hot spots of the flow cytometry detection device in the embodiment of the present invention are to biology
The temporal summation of the irradiation of particle increases, and single hot spot constant or even more existing skill possible to the irradiation time of biological particle
Art is shortened, and therefore, is formed at least two hot spots on F-L curve and has been taken into account to the detectability of small-signal and to list
The resolving power of a biological particle, Flow cytometry device list particle resolving power that the embodiment of the present invention is proposed and to faint
The detection power of signal is strong.Hot spot formation component is formed by least two hot spot relative positions and fixes, and then the embodiment of the present invention
The Flow cytometry device proposed can be realized the Accurate Determining to biological particle speed.
Specifically, according to an embodiment of the invention, it is binary optical device that the hot spot, which forms component,.Binary optical device
Micro-structure can be changed incident laser each point at light path, specific optical diffraction occurs, so can be formed on output plane to
Few two hot spots.Binary optical device is formed by optical power detection inside hot spot, thus even if biological particle passes through hot spot
Relative position when certain deviation occurs, irradiation condition that particle is subject to is protected substantially without optical signalling of significant change, its output
Hold it is constant, thus improve the stability of detection, reduce detection signal the coefficient of variation；The boundary of hot spot is sharp keen, so that defeated
Optical signalling out has clearly boundary, helps to identify the optical signal that biological particle issues；Intense part inside hot spot
Cloth is uniform, and output optical signal amplitude when this makes biological particle pass through rectangular light spot is held essentially constant, convenient for optical signal
It is calculated, such as calculates height, width or the area of optical signal.When forming more than two hot spots, intermediate hot spot is for increasing
To the irradiation time of biological particle, extend duration of optical signal, convenient for being collected to optical signal, detection and analysis, mentions
High detection sensitivity, is particularly helpful to realize effective detection to faint optical signal.It is binary optical device that hot spot, which forms component,
Part makes the detection stability of the Flow cytometry device of the embodiment of the present invention, single particle resolving power and examines to small-signal
Survey ability further increases.
Specifically, according to an embodiment of the invention, at least two hot spot is separately rectangle.It is according to the present invention
Embodiment, the Energy distribution in rectangular light spot is uniform and boundary is sharp keen, and then biological particle is sent out by the relative position of hot spot
When raw change, the optical signalling of output will not change therewith, so that rectangular light spot makes the streaming of the embodiment of the present invention
The detection stability of cell art detection device, single particle resolving power and high speed and high-throughput detection efficiency further increase.And show
Have in technology, by the elliptical spot of lens forming, light intensity is not boundary consecutive variations, sharp keen at Gaussian Profile, thus its
The fluorescence signal of excitation has significant difference by the difference of position because of biological particles such as cells, and signal is in approximate Gauss
Shape is unfavorable for differentiating the signal of the biological particles such as individual cells, reduces sensitivity and resolving power.
According to an embodiment of the invention, at least two rectangular light spot intervals are arranged, and the central axes of the rectangle are located at
On the F-L curve.Wherein, the central axes of rectangle and the long side of rectangle are vertical, parallel with the short side of rectangle.The present invention is implemented
The sample flow comprising biological particles such as cells of example is focused on the center of runner, and liquid stream on the center of runner
Flow velocity highest, thus hot spot central axes are overlapped with F-L curve individual cells can be made to shorten by time of hot spot, both single
In the time of position cell flux can be improved by more cells.The long side of rectangular light spot and the central axes of rectangle are vertical,
It is i.e. vertical with F-L curve namely vertical with cell flow direction, it so still can be by light when cell deviates F-L curve position
Effective detection is realized in spot irradiation.In turn, the central axes of the rectangular light spot of the embodiment of the present invention are located on the F-L curve,
So that the detection flux of Flow cytometry device and sensitivity further increase.
According to an embodiment of the invention, the long side length of at least two rectangular light spots is 10~100 microns, it is described at least
The bond length of the both ends hot spot of two rectangular light spots is the size determination based on sample to be tested.Wherein, both ends hot spot refers to,
When tool is there are two hot spot, both ends hot spot is as shown in 11, the 12 of Fig. 1, when with more than two hot spots, such as three hot spots, two
Hold hot spot as shown in 51, the 52 of Fig. 6.Meanwhile when there are two above rectangular light spots, such as three hot spots, as shown in fig. 6,53 quilts
Referred to as intermediate hot spot.In the present invention, the long side of both ends rectangular light spot refers to the slightly long side of length, the long side of intermediate rectangular hot spot
Refer to the side vertical with F-L curve.The long side length of rectangular light spot, which is 10~100 microns, to be avoided to greatest extent to biology
The missing inspection of particulate samples.The bond length of both ends hot spot is the size determination based on sample to be tested, the dress of the embodiment of the present invention
The bond length for the both ends hot spot set and then can further improve to single biological particle close to the average value of sample to be tested size
The resolving power of sample.
According to an embodiment of the invention, the both ends hot spot of at least two hot spots is respectively provided with identical size, and institute
The long side length for stating at least two hot spots is equal.In turn, the light intensity of both ends hot spot is equal, and biological particle passes through at least two
The amplitude that optical signal is generated after hot spot is of substantially equal (it should be noted that the light intensity of hot spot described in this specification is equal is
Refer to it is approximately equal, rather than it is essentially equal.Because binary optical device can only theoretically realize it is essentially equal, in practice not
In the presence of).And then the accuracy of test can be improved by data processing methods such as weighted averages in data handling.
Meanwhile according to an embodiment of the invention, the amplitude mean square deviation of at least two hot spots less than 10%.The embodiment of the present invention
In, for the amplitude mean square deviation of at least two hot spots less than 10%, the light intensity of hot spot is equal, then biological particle sample to be tested is by least
The amplitude that optical signal is generated after two hot spots is of substantially equal, can be mentioned in data handling by data processing methods such as weighted averages
The accuracy of height test.
According to an embodiment of the invention, the uniformity of the light intensity of at least two hot spots is higher than 80%, light distribution is uniform, because
And when biological particle passes through at least two hot spots, even if relative position changes, excitation laser suffered by biological particle is had no
The optical signalling of significant difference, output will not change therewith, so that signal difference caused by positional factor is reduced, and
And the rising edge and failing edge of the optical signal generated are more steep, top is more smooth, and then are conducive to know optical signal
It not and handles, further improves stability, accuracy and the sensitivity of Flow cytometry device.
More specifically, according to an embodiment of the invention, the Flow cytometry device, with reference to Figure 12, comprising: along
The optical path of incident laser is disposed with laser 100, beam expanding lens 200, the hot spot and forms component 300, the first aperture
1400 and first condenser lens 400, the laser that laser 100 is launched component 300 is formed on 10 middle line of runner by hot spot
At least two hot spots are formed, wherein runner 10 can be transparent fluid channel, can also be and is present in air, wherein aperture
The 1400 each secondary formed except the defeated diffraction pattern main of component 300 for removing hot spot.When sample (cell or other lifes to be measured
Object particle) when passing through hot spot along 10 middle line of runner, sample is inspired fluorescence, forward scattering light (forward scattered light) and lateral dissipates
Light is penetrated, along the optical path of forward scattering laser, is disposed with light barrier 1800, the first optical filtering 500 and the first photoelectric sensing
Device 600, light barrier 1800 are located at the optical path Center of forward scattering light, and area is small, can block laser directly incoming signal, protect
It is only scattered light signal that card, which is incident on the signal of photoelectric sensor 600, and the first optical filtering 500 can make to be incident on photoelectric sensor
600 signal is only laser signal, and the first photoelectric sensor 600 is used to convert electric signal for forward-scattering signal, carry out
Optical signal analysis, and then analyze sample message；The laterally optical path of scattering laser is disposed with 700 He of the second optical filtering
Second photoelectric sensor 800, the second optical filtering 700 can make the signal for being incident on photoelectric sensor 800 be only laser signal, and second
Photoelectric sensor 800 is used to convert lateral scattering optical signal to electric signal, carries out optical signal analysis, and then analyzes sample letter
Breath；Along the optical path for the fluorescence that is excited, it is disposed with the second condenser lens 1700 and multiplex fluorescence detection device 2000, second
Condenser lens 1700 is for the fluorescence in collection space, to enhance fluorescence signal, wherein the multiplex fluorescence detection device
2000 include dichroscope 900, second orifice diaphragm 1500, third optical filtering 1000 and third photoelectric sensor 1100, two to
Look mirror 900 is used to multicolor fluorescence being separated into one-color fluorescence, i.e., the separation of every color fluorescence signal be both needed to a dichroscope 900 into
Row separation, and then every color fluorescence passes sequentially through second orifice diaphragm 1500, third optical filtering 1000 and third photoelectric sensor
1100, dichroscope 900, second orifice diaphragm 1500, third optical filtering 1000 and third photoelectric sensor 1100 herein be
Serial dichroscope, serial second orifice diaphragm, serial third optical filtering and serial third photoelectric sensor general designation (with reference to figure
12), wherein second orifice diaphragm 1500 is used to retain the fluorescence of particular color for removing stray light, third optical filtering 1000
Signal, third photoelectric sensor 1100 is used to convert the fluorescence signal of particular color to electric signal, and then carries out optical signal point
Analysis, to obtain sample message.Wherein, the intensity of fluorescence signal and the surface antigen quantity of biological particle are positively correlated, and forward direction dissipates
The size of the intensity and biological particle of penetrating light is positively correlated, the intensity of side scattered light and surface granularity, the internal junction of biological particle
The oneself factor of the biological particles such as structure, optical characteristics is related.According to an embodiment of the invention, photoelectric sensor includes but is not limited to
Photodiode, photomultiplier tube, photodiode array and multiplier tube array, wherein scattering light is mostly used photodiode, glimmering
Light is mostly used photomultiplier tube or photodiode array or multiplier tube array.And along observation optical path, it is disposed with image
Sensor 1200, the 4th optical filtering 1600 and tertiary focusing lens 1300, imaging sensor 1200 for the sample in runner into
Row observation in real time and monitoring, to facilitate experimental implementation, the 4th optical filtering 1600 is used to remove the interference of laser signal.The present invention is real
The Flow cytometry device of example is applied under said modules composition, easy to operate, stability, the accuracy, Dan Wei of detection
Grain resolving power and high speed and high-throughput detection efficiency significantly improve.
Flow cytometry assays
In the second aspect of the present invention, the invention proposes a kind of flow cytometry assays.Reality according to the present invention
Example is applied, which includes: that at least two hot spots are formed on F-L curve, wherein at least two hot spot is as preceding
It is defined.The flow cytometry assays that the embodiment of the present invention is proposed can Accurate Determining biological particle sample to be tested speed
The stability of degree, testing result is high, can take into account single particle resolving power and the detection to small-signal.
Specifically, according to an embodiment of the invention, this method be using mentioned-above Flow cytometry device into
Capable.As previously mentioned, the Flow cytometry device of the embodiment of the present invention has testing result stability height, to single particle
Resolving power is strong and the feature strong to the detectability of small-signal, and then the detection method of the embodiment of the present invention is using the present invention
The Flow cytometry device of embodiment detects biological particle sample to be tested, the stability of testing result, to list
The resolving power of particle and the detectability of small-signal is further increased.
Purposes of the binary optical device in Flow cytometry or preparation Flow cytometry device
In the third aspect of the present invention, the invention proposes binary optical devices in Flow cytometry or preparation streaming
Purposes in cell art detection device.According to an embodiment of the invention, binary optical device can according to the mode of incident laser, swash
Wavelength, the diameter of light design corresponding binary optical device for different incident lasers, to apply in flow cytometry
In detection or preparation Flow cytometry device, at least two hot spots, and then binary optical device are formed on F-L curve
Apply in Flow cytometry or preparation Flow cytometry device, the detection method or device can Accurate Determining biology it is micro-
The speed of grain, resolving power to single particle and detection energy to small-signal high to the stability of the testing result of biological particle
Power is high.
According to a particular embodiment of the invention, the binary optical device is modulated the phase of incident laser, in turn
At least two hot spots are being formed on F-L curve, the constant distance of at least two hot spots, boundary is sharp keen, and internal light distribution is equal
It is even, single particle resolving power and the detection to small-signal can be taken into account, and then using binary optical device to the phase of incident laser
Be modulated, the velocity determination accuracy of biological particle further increased, stability to the testing result of biological particle and
Resolving power to single particle and the detectability of small-signal is further increased.
In addition, according to an embodiment of the invention, the material of the binary optical device is selected from quartz, glass, poly- methyl-prop
At least one of e pioic acid methyl ester.The selection of the material of binary optical device is to carry out selection for different incident lasers, this
The material of the binary optical device of inventive embodiments is selected from but not limited to quartz, glass, polymethyl methacrylate, as long as can be
At least two hot spots are formed on F-L curve, at least two hot spot is for another example described above.Select most suitable material
Material prepares binary optical device, can further improve the stability of the Flow cytometry or Flow cytometry device
And sensitivity.
In addition, according to an embodiment of the invention, the laser of the embodiment of the present invention include but is not limited to Gauss light, planar light and
The binary optical device of multimode light, the embodiment of the present invention can be designed according to different incident lasers, and then in F-L curve
At least two hot spots required for upper formation is tested.Suitable incident laser and its corresponding binary optical device are selected, it can be into
One step significantly improves the stability and sensitivity of the Flow cytometry or Flow cytometry device.
According to a particular embodiment of the invention, when the laser is the Gauss light that diameter is 3mm, the binary optical device
The thin slice that the structure of part is square, the side length of the square is 3mm and inside has 128*128 step.The present invention is real
The structure for applying the above-mentioned specific binary optical device of example can form two or three rectangular light spots, the square on F-L curve
Shape hot spot has the characteristics that at least one of mentioned-above hot spot, the structure of the above-mentioned binary optical device of the embodiment of the present invention
It further improves the stability of the flow cytometry assays or Flow cytometry device and single particle is divided
Distinguish power and the detectability to small-signal.
The embodiment of the present invention is described below in detail.The embodiments described below is exemplary, and is only used for explaining this hair
It is bright, and be not considered as limiting the invention.Particular technique or condition are not specified in embodiment, according to text in the art
It offers described technology or conditions or is carried out according to product description.Reagents or instruments used without specified manufacturer,
For can be with conventional products that are commercially available.
Embodiment 1
In the present embodiment, inventor forms two rectangular light spots using binary optical device on F-L curve, below
It is the description of the feature and design to rectangular light spot:
As shown in Figure 1, double square hot spot 1, two rectangular light spots 11 and 12 are separated by a certain distance L, two rectangular light spots
Long side and bond length are respectively l1 and w1, l2 and w2, the optical power detection in hot spot.
When Flow cytometry, the positional relationship situation between hot spot, biological particle and runner is as shown in Figure 2: light
Spot 1 is located at the center of runner, i.e. the middle line and F-L curve 43 of rectangular light spot long side is overlapped, and F-L curve 43 is located at runner
The center on boundary 41 and 42；The flow direction 3 of biological particle 2 is parallel to F-L curve 43, and as close as possible to middle line.General biology
Particle is near F-L curve in the range of more than ten microns, thus it is tens that inventor, which takes two rectangular light spot long sides l1 and l2,
Micron, to prevent the missing inspection to biological particle.Go out single biological particle for fine-resolution, inventor is short by two rectangular light spots
Side w1 and w2 are adjusted to the characteristic size close to single biological particle.
Biological particle by the optical signal that is issued when double square hot spot as shown in figure 3, optical signal here can be referred to it is preceding to
Scattered light signal, lateral scattering optical signal or certain fluorescence signal all the way, twice the time interval of optical signal be T, amplitude H1
With H2, duration be T1 and T2, signal are A1 to the integral of time and A2 (i.e. area).Inventor has found that hot spot boundary is clear
Clear, internal optical power detection, the rising edge and failing edge that biological particle passes through the optical signal generated when hot spot are more steep, push up
Portion is more smooth, this is conducive to identify optical signal and handled.Pass through the time interval of two hot spots by biological particle
T can obtain the flow velocity V=L/T of biological particle.The amplitude and area of optical signal are used to characterize the intensity of optical signal, wherein fluorescence
The intensity of signal and the surface antigen quantity of biological particle are positively correlated, the intensity of forward scattered light and the size positive of biological particle
It closes, the oneself factor of the biological particles such as the surface granularity of the intensity of side scattered light and biological particle, internal structure, optical characteristics
It is related.For convenient for signal processing, inventor chooses the equal sized equal with light intensity of two rectangular light spots.Biological particle is logical in this way
The optical signal for crossing the generation of two rectangular light spots is of substantially equal, in data handling can be by taking the data processing methods such as average
The accuracy of test is improved, for example, showing that the average value of signal is (H1+H2)/2, (T1+T2)/2 and (A1+A2)/2.
Inventor is described specifically the parameter and design of double square hot spot using haemocyte as biological particle to be checked:
It mainly include red blood cell, leucocyte and blood platelet in blood, red blood cell is recessed dish, diameter is 7~8 microns, thickness 2
~2.5 microns, leucocyte diameter is mostly 10~15 microns, and blood platelet is convex dish, 2~3 microns of diameter.Can accurately to differentiate
The minor axis length of rectangular light spot is designed as 10 microns by the optical signal of a haemocyte out, inventor.Generally, biological particle is flowing
Road middle line is nearby in the range of more than ten microns, thus the long side length of rectangular light spot is set as 50 microns by inventor, to prevent pair
The missing inspection of biological particle.In addition, the flow velocity of biological particle is mostly 3~10m/s, and then inventor is by the spacing of two rectangular light spots
100 microns are designed as, it is larger by the time interval of two rectangular light spots to guarantee haemocyte, to improve to haemocyte flow velocity
The accuracy of measurement.For example, the time interval of two optical signals in the same channel that haemocyte is issued by two hot spots is 2
Microsecond, it is concluded that its flow velocity be 5m/s.
To realize this hot spot, inventor is modulated the phase of incident laser using binary optical device, that is, utilizes
Micro- step structure on binary optical device surface changes the light path at incident laser each point, modulated laser by lens and
Expectation hot spot is realized on its focal plane.Binary optical device is made of transparent optical material, such as quartz, glass, PMMA, is led to
The methods of over etching, focusing plasma art processing produce the step of different height on the surface of optical material.Incident laser can
For the laser or even multimode light of Gauss light, planar light or other modes, the changeable parameters such as wavelength, the diameter of laser, for difference
Incident laser, can be designed corresponding binary optical device.Such as: it is formed by for realization is above-mentioned by two rectangular light spots
Binary optical device can be prepared into the pros of side length 3mm when selecting the Gauss light of cut-off diameter 3mm as input laser by hot spot
Shape thin slice, inside be divided into 128 × 128 steps, the phase diagram of this binary optical device is as shown in Figure 4.Inventor is further
The focal length of lens after choosing binary optical device is 10mm, obtains the output light distribution on focal plane as shown in figure 5, black in figure
Region indicates that light intensity is zero, and Pure White indicates that light intensity is maximum, and grey expression falls between.Inventors have found that above-mentioned two
The size of the output facula of first optical device is consistent with design, has clearly boundary, and the amplitude mean square deviation in hot spot is less than
10%, with the uniformity of height.
Embodiment 2
In the present embodiment, inventor forms three rectangular light spots using binary optical device on F-L curve, below
It is the description of the feature and design to rectangular light spot:
As shown in fig. 6, rectangular light spot 51 and 52, Yi Jiwei of the hot spot 5 formed by three rectangles by two distance L
Rectangular light spot 53 among the two collectively constitutes, and size of the intermediate rectangular light spot on biological particle flow direction is bigger,
The size of three rectangles is respectively w1 × l1, w2 × l2 and w3 × l3, the optical power detection in hot spot.
When Flow cytometry, the positional relationship situation between hot spot, biological particle and runner is as shown in Figure 7: light
Spot is located at the center of liquid stream, and both the common middle line of three rectangles was overlapped with F-L curve 43, and F-L curve 43 is located at runner
The center on boundary 41 and 42；The flow direction 3 of biological particle 2 is parallel to F-L curve 43, and as close as possible to middle line.The square at both ends
Shape hot spot 51 and 52 is for differentiating single biological particle and testing the speed to it, and intermediate wide rectangle 53 is for increasing to biology
The irradiation time of particle, the duration for both having extended optical signal improve convenient for being collected to optical signal, detection and analysis
Detection sensitivity is particularly helpful to realize effective detection to faint optical signal.General biological particle is ten near F-L curve
In the range of several microns, and then the length adjustment of long side l1, l2 and l3 of rectangular light spot are tens microns by inventor, to prevent
Missing inspection to biological particle.Go out single biological particle for fine-resolution, inventor is by the short side w1 and w2 of rectangular light spot 51 and 52
It is adjusted to the characteristic size close to single biological particle.
Biological particle by the optical signal that is issued when three rectangular light spots as shown in figure 8, optical signal here can be referred to it is preceding to
Scattered light signal, lateral scattering optical signal or certain fluorescence signal all the way, pass through the time interval of both ends rectangular light spot 51 and 52
For T, optical signal amplitude is H1, H2 and H3, duration T1, T2 and T3, signal to the integral of time for A1, A2 and A3 (i.e.
Area).Inventors have found that hot spot sharpness of border, internal optical power detection, biological particle pass through the optical signal generated when hot spot
Rising edge and failing edge is more steep, top is more smooth, this is conducive to identify optical signal and handled.Pass through calculating
Biological particle obtains the flow velocity V=L/T of biological particle by the time interval T of both ends rectangular light spot 51 and 52.It is given birth to by calculating
Object particle passes through the area (i.e. the time integral of optical signal) of 53 optical signal of intermediate rectangular hot spot, can effectively enhance to faint
The detectability of optical signal.The amplitude and area of optical signal are used to characterize the intensity of optical signal, wherein the intensity of fluorescence signal with
The surface antigen quantity of biological particle is positively correlated, and the intensity of forward scattered light and the size of biological particle are positively correlated, lateral scattering
The oneself factors of biological particles such as the intensity of light and the surface granularity of biological particle, internal structure, optical characteristics are related.For convenient for
Signal processing, the light intensity that inventor designs three hot spots is equal, side length l1, l2 and l3 phase of three rectangular light spots perpendicular to flow direction
Deng the short side w1 and w2 of rectangular light spot 51 and 52 are equal.The biological particle amplitude that passes through the optical signals of three rectangular light spots in this way
It is of substantially equal, the accuracy of test can be improved by data processing methods such as weighted averages in data handling in this way.Such as:
, there are T1:T2:T3 ≈ w1:w2:w3, A1:A2:A3 ≈ T1:T2:T3 ≈ in H1:H2:H3 ≈ 1:1:1 when light intensity is equal, when flow speed stability
W1:w2:w3, the weighted average of optical signal amplitude are (H1 × w1+H2 × w2+H3 × w3)/(w1+w2+w3) or (H1 × T1+
H2 × T2+H3 × T3)/(T1+T2+T3) and signal mean value (H1+H2)/2, (H1+H2+H3)/3, (T1+T2)/2 and (A1+
A2)/2。
Inventor is described specifically the parameter and design of three rectangular light spots using haemocyte as biological particle to be checked:
It mainly include red blood cell, leucocyte and blood platelet in blood, red blood cell is recessed dish, diameter is 7~8 microns, thickness 2
~2.5 microns, leucocyte diameter is mostly 10~15 microns, and blood platelet is convex dish, micro- 2~3 microns of diameter.Can accurately to divide
The optical signal of a haemocyte is discerned, the length adjustment of the short side w1 and w2 of both ends rectangular light spot 51 and 52 are 10 micro- by inventor
Rice.To increase the time that haemocyte passes through intermediate rectangular hot spot, inventor is by intermediate rectangular hot spot 53 in biological particle flowing side
Upward side length w3 is adjusted to 50 microns.Generally, biological particle more than ten microns of region near F-L curve, inventor is by three
A rectangular light spot is 50 microns perpendicular to the length adjustment of side length l1, l2 and l3 of flow direction, to prevent the missing inspection to biological particle.
In addition, the flow velocity of biological particle stream is mostly 3~10m/s, the spacing of two narrow rectangular light spots at both ends is adjusted to 100 by inventor
Micron measures haemocyte flow velocity to improve with guaranteeing that haemocyte is sufficiently large by the time interval of two rectangular light spots of person
Accuracy.For example, two optical signals in the same channel that two hot spots 51 and 52 that biological particle passes through both ends are issued
Time interval is 2 microseconds, it is concluded that its flow velocity is 5m/s.
To realize this hot spot, inventor is modulated the phase of incident laser using binary optical device, that is, utilizes
Micro- step structure on binary optical device surface changes the light path at incident laser each point, modulated laser by lens and
Expectation hot spot is realized on its focal plane.Binary optical device can be used transparent optical material and be made, such as quartz, glass, PMMA,
The step of different height is produced on the surface of optical material by etching, focusing the methods of plasma art processing.Incident laser
It can be Gauss light, the laser of planar light or other modes or even multimode light, the changeable parameters such as wavelength, the diameter of laser, for not
Same incident laser, can be designed corresponding binary optical device.Such as: it is formed by realization is above-mentioned by three rectangular light spots
Hot spot, when select cut-off diameter be 3mm Gauss light as input laser when, can by binary optical device be fabricated to side length 3mm
Square sheets, inside be divided into 128 × 128 steps, the phase diagram of corresponding binary optical device is as shown in Figure 9.Invention
It is 10mm that person, which takes the focal length of lens after binary optical device, show that the output light distribution on focal plane is as shown in Figure 10, in figure
Black region indicates that light intensity is zero, and Pure White indicates that light intensity is maximum, and grey expression falls between；On inventors have found that
The size for stating the output facula of binary optical device is consistent with design, has clearly boundary, the amplitude mean square deviation in hot spot
Less than the 15%, uniformity with height.
Embodiment 3
In the present embodiment, the optical path of inventor's flow cytometric detection device is described in detail:
With reference to Figure 11, laser 61 reaches the design bore of binary optical device after beam expander 200 expands, and laser passes through
Hot spot is focused using aperture 1400 by lens 400 after forming component 300 (this embodiment is binary optical device) shaping
At the center of runner 10, the biological particle at runner center is illuminated, constitutes hot spot molding optical path above.Life in runner
Object particle generates fluorescence and scattered light signal when passing through hot spot, optical signal is detected optical path detection.Fluorescence and scattered light signal are logical
It crosses light path to be separated one by one, such as forward scattering light 63, side scattered light 62 excite the fluorescence 131 generated and by color separation
Assorted 161~the 16N of fluorescence obtained afterwards, each road optical signal are converted by photoelectricity testing parts such as photomultiplier tube, photodiodes
For electric signal, electric signal completes signal processing on subsequent processing platform.It is convenient for debugging, figure can also be added in light path
As sensor 1200 and the second condenser lens 1300 and optical filtering 1600 realize that light field 21 is observed.
In the description of the present invention, it is to be understood that, term " center ", " longitudinal direction ", " transverse direction ", " length ", " width ",
" thickness ", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom" "inner", "outside", " up time
The orientation or positional relationship of the instructions such as needle ", " counterclockwise ", " axial direction ", " radial direction ", " circumferential direction " be orientation based on the figure or
Positional relationship is merely for convenience of description of the present invention and simplification of the description, rather than the device or element of indication or suggestion meaning must
There must be specific orientation, be constructed and operated in a specific orientation, therefore be not considered as limiting the invention.
In addition, term " first ", " second " are used for descriptive purposes only and cannot be understood as indicating or suggesting relative importance
Or implicitly indicate the quantity of indicated technical characteristic.Define " first " as a result, the feature of " second " can be expressed or
Implicitly include one or more of the features.In the description of the present invention, the meaning of " plurality " is two or more,
Unless otherwise specifically defined.
In the present invention unless specifically defined or limited otherwise, term " installation ", " connected ", " connection ", " fixation " etc.
Term shall be understood in a broad sense, for example, it may be being fixedly connected, may be a detachable connection, or integral；It can be mechanical connect
It connects, is also possible to be electrically connected；It can be directly connected, can also can be in two elements indirectly connected through an intermediary
The interaction relationship of the connection in portion or two elements.It for the ordinary skill in the art, can be according to specific feelings
Condition understands the concrete meaning of above-mentioned term in the present invention.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
It must be directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be in office
It can be combined in any suitable manner in one or more embodiment or examples.In addition, without conflicting with each other, the skill of this field
Art personnel can tie the feature of different embodiments or examples described in this specification and different embodiments or examples
It closes and combines.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (12)
1. a kind of Flow cytometry device characterized by comprising
Binary optical device, the laser that the binary optical device is configured as being suitable for launching single laser is in runner
At least two hot spots are formed on line, at least two hot spot is separately rectangle, the light intensity of at least two hot spot
Uniformity be higher than 80%；
Wherein, along the optical path of incident laser, laser, beam expanding lens, the binary optical device, the first aperture are disposed with
Diaphragm and the first condenser lens,
Along the optical path of forward scattering laser, it is disposed with light barrier, the first optical filtering and the first photoelectric sensor,
The laterally optical path of scattering laser is disposed with the second optical filtering and the second photoelectric sensor,
Along the optical path for the fluorescence that is excited, it is disposed with the second condenser lens and multiplex fluorescence detection device, wherein described more
Road fluorescence detection device includes dichroscope, second orifice diaphragm, third optical filtering and third photoelectric sensor, and
Along observation optical path, it is disposed with image optical flame detector, the 4th optical filtering and tertiary focusing lens.
2. Flow cytometry device according to claim 1, which is characterized in that at least two spot separation is set
It sets, and the central axes of the rectangle are located on the F-L curve.
3. Flow cytometry device according to claim 2, which is characterized in that the long side of at least two hot spot
Length is 10~100 microns, and the bond length of the both ends hot spot of at least two hot spot is the size based on sample to be tested
Determining.
4. Flow cytometry device according to claim 1, which is characterized in that the both ends of at least two hot spot
Hot spot is respectively provided with identical size, and the long side length of at least two hot spot is equal.
5. Flow cytometry device according to claim 1, which is characterized in that the amplitude of at least two hot spot
Mean square deviation is less than 10%.
6. a kind of flow cytometry assays characterized by comprising
At least two hot spots are formed on F-L curve,
Wherein, at least two hot spot is as defined in any one of Claims 1 to 5.
7. according to the method described in claim 6, it is characterized in that, the flow cytometry assays are to utilize claim
What 1~5 described in any item Flow cytometry devices carried out.
8. purposes of the binary optical device in Flow cytometry or preparation Flow cytometry device, the binary optical
Learn the laser that device is configured as being suitable for launching single laser and form at least two hot spots on F-L curve, it is described extremely
Few two hot spots are separately rectangle, and the uniformity of the light intensity of at least two hot spot is higher than 80%；
Wherein, the structure of the Flow cytometry device are as follows:
Along the optical path of incident laser, it is disposed with laser, beam expanding lens, the binary optical device, the first aperture
With the first condenser lens,
Along the optical path of forward scattering laser, it is disposed with light barrier, the first optical filtering and the first photoelectric sensor,
The laterally optical path of scattering laser is disposed with the second optical filtering and the second photoelectric sensor,
Along the optical path for the fluorescence that is excited, it is disposed with the second condenser lens and multiplex fluorescence detection device, wherein described more
Road fluorescence detection device includes dichroscope, second orifice diaphragm, third optical filtering and third photoelectric sensor, and
Along observation optical path, it is disposed with image optical flame detector, the 4th optical filtering and tertiary focusing lens.
9. purposes according to claim 8, which is characterized in that the binary optical device carries out the phase of incident laser
Modulation.
10. purposes according to claim 8, which is characterized in that the material of the binary optical device is selected from quartz, glass
At least one of glass, polymethyl methacrylate.
11. purposes according to claim 9, which is characterized in that the laser includes being selected from Gauss light, planar light and multimode
At least one of light.
12. purposes according to claim 9, which is characterized in that when the laser is the Gauss light that diameter is 3mm, institute
The structure for stating binary optical device is square, and the side length of the square is 3mm and inside has 128*128 step.
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| CN111855544B (en) * | 2020-07-31 | 2021-11-26 | 上海微电子装备（集团）股份有限公司 | Fluorescence imaging device and imaging method thereof |
| CN113624666B (en) * | 2021-09-07 | 2022-09-23 | 清华大学 | Stream type imaging system based on dot matrix laser scanning |
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| CN101943663B (en) * | 2010-07-09 | 2012-07-25 | 董珂 | Measuring analytical system and measuring analytical method for distinguishing diffraction image of particles automatically |
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| US9103760B2 (en) * | 2012-06-09 | 2015-08-11 | E. I. Spectra, Llc | Fluorescence flow cytometry device and method |
| CN202886734U (en) * | 2012-11-19 | 2013-04-17 | 上海高意激光技术有限公司 | Polychromatic optical system for flow cytometry |
| CN103852409B (en) * | 2014-03-18 | 2016-09-14 | 江西科技师范大学 | The imaging system of hemocyte in flow cytometer |
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